DIFFERENTIAL FUNCTION AND REGULATION OF RUNX2 ISOFORMS

描述（由申请人提供）：复杂的Cbfa1/Runx2基因产生两个异构体，Runx2- ii和Runx2- i，分别来自远端“骨相关”（P1）和近端（P2）启动子。II型和I型Cbfa1基因产物除了其5‘非翻译区（5’ utr）和n端不同外，是完全相同的。这些异构体在包括成骨、软骨和牙形成在内的发育途径中赋予不同功能的分子机制仍有待确定，但可能来自不同的n端功能域，并可能由其5' utr调节翻译效率的能力所赋予的差异表达。Runx2-II II n端异构体在骨骼发育过程中的后期表达表明其具有独特的功能，但之前删除这两个异构体的转基因研究并没有设计用于评估其单独的功能，此外，Runx2基因产生多个具有5' utr的mrna，具有复杂的二级结构，通过内部核糖体进入位点（IRES）启动蛋白质合成来调节体外翻译。这些研究的总体目标是建立Runx2-II和Runx2-I异构体的不同功能，并确认ires依赖的翻译控制在体内调节其表达和组织特异性功能中起重要作用。在目标1中，我们将通过完成Runx2-II亚型选择性缺陷小鼠的表征来确定“骨相关”亚型的单独功能。此外，我们将创建一个选择性Runx2-I空鼠标，使Runx2-II异构体的表达和功能保持完整。在目标2中，我们将在体外和转基因小鼠中研究I型和Il - Cbfa1的5' utr通过cap和ires依赖机制调节翻译效率的能力。这些研究将定义n端结构域的独特功能，并确定翻译调控是否有助于Runx2-I和Runx2-II亚型的组织特异性表达。

DESCRIPTION (provided by applicant): The complex Cbfa1/Runx2 gene gives rise to two isoforms, Runx2-II and Runx2-I, respectively derived from the distal "bone-related" (P1) and proximal (P2) promoters. Except for differences in their 5' untranslated regions (5'UTRs) and N-termini, the Type II and I Cbfa1 gene products are identical. The molecular mechanisms whereby these isoforms impart differential function to developmental pathways involving osteogenesis, chondrogenesis, and odontogenesis remain to be defined, but are likely derived from distinct N-terminal functional domains and possibly differential expression imparted by the ability of their 5'UTRs to modulate translational efficiency. Distinct function of the Runx2-II II N-terminal isoform is suggested by its later expression during bone development, but prior transgenic studies that deleted both isoforms were not designed to evaluate their separate function, in addition, the Runx2 gene generates multiple mRNAs with 5'UTRs having complex secondary structures that regulate translation in vitro through internal ribosome entry site (IRES) initiation of protein synthesis. The overall goals of these investigations are to establish the distinct function of Runx2-II and Runx2-I isoforms and to confirm that IRES-dependent translation control plays an important role in regulating its expression and tissue specific functions in vivo. In aim 1, we will determine the separate function of the "bone-related" isoform by completing the characterization of mice selectively deficient in the Runx2-II isoform. In addition, we will create a selective Runx2-I null mouse that leaves the expression and function of the Runx2-II isoform intact. In aim 2, we will investigate the ability of the 5'UTRs of Type I and Il Cbfa1 to regulate translation efficiency through cap-and IRES-dependent mechanisms in vitro and in transgenic mice. These studies will define the unique functions of the N-terminal domains, and determine if translational regulation contributes to the tissue-specific expression of Runx2-I and Runx2-II isoforms.